K14 mRNA reprogramming for dominant epidermolysis bullosa simplex

Abstract

The major challenge to a successful gene therapy of autosomal dominant genetic diseases is a highly efficient and specific knock-down or repair of the disease-causing allele. In epidermolysis bullosa simplex-type Dowling-Meara (EBS-DM), a single amino acid exchange in exon 1 of the keratin 14 gene (K14) triggers a severe skin phenotype, characterized by blistering of the skin and mucous membranes after minor trauma. We chose spliceosome-mediated RNA trans-splicing to specifically replace exons 1-7 of the K14 gene. In this approach, the mutated coding region is replaced by an RNA-trans-splicing molecule (RTM) that incorporates a binding domain (BD) and the wild-type sequence of K14. Since the BD is crucial for the trans-splicing functionality, we developed a fluorescence-based RTM screen consisting of an RTM library containing random BDs. Co-transfection of the library with a target molecule enabled us to identify highly functional RTMs. The best RTMs were adapted for endogenous trans-splicing in an EBS-DM patient cell line. In this cell line, we were able to detect functional, efficient and correct trans-splicing on RNA and protein levels. Scratch assays confirmed phenotypic reversion in vitro. Owing to concomitant knock-down and repair of the mutated allele, we assume that trans-splicing is a promising tool for the treatment of autosomal dominant genetic disease.

Development of Allele-Specific Therapeutic siRNA for Keratin 5 Mutations in Epidermolysis Bullosa Simplex

“…An alternative means of treating dominant keratin mutations is by gene correction via trans-splicing, although low correction frequency remains an issue for this method (Wally et al, 2010). However, dominant-negative disorders such as EBS also lend themselves to therapy based on small interfering RNA (siRNA), as reviewed in Lane and McLean (2008) and Leachman et al (2008).…”

High-Throughput Screening for Highly Functional RNA-Trans-Splicing Molecules: Correction of Plectin in Epidermolysis Bullosa Simplex

“…Several approaches in different settings (i.e. reporter gene based, endogenous trans-splicing, in vivo application) have www.intechopen.com corroborated this assumption (Dallinger et al, 2003;Coady and Lorson, 2010;Wally et al, 2010). Also the applicability of SMaRT beyond mRNA repair was shown, using it for in vivo imaging and antibody and protein production.…”

“…Such settings were reported for epidermolysis bullosa (COL7A1, KRT14, PLEC) (Murauer et al, 2010;Wally et al, 2010;Wally et al, 2008), Duchenne muscular dystrophy (Lorain et al, 2010), cystic fibrosis (Liu et al, 2002;Song et al, 2009), frontotemporal dementia with parkinsonism (Rodriguez-Martin et al, 2009), severe combined immunodeficiency (Zayed et al, 2007), spinal muscular atrophy (Coady et al, 2007), sickle cell anemia and ß-thalassemia (Kierlin-Duncan and Sullenger, 2007). First in vivo assays showed the functionality of SMaRT in a mouse model of spinal muscular atrophy (Coady et al, 2008;Coady and Lorson, 2010).…”

“…For the latter, therapeutic protein encoding sequences were trans-spliced to the highly abundant albumin mRNA, resulting in the expression of the therapeutic molecule (Wang et al, 2009). For epidermolysis bullosa, SMaRT was applied for a number of underlying genes, including PLEC (Wally et al, 2008), COL7A1 (Murauer et al, 2010), K14 (Wally et al, 2010) and COL17A1 (our unpublished results). Whereas for PLEC and COL7A1 the binding domains of the RNA-trans-splicing molecules (RTMs) were designed empirically, based on preliminary data, the binding domains for K14 and COL17A1 resulted from the evaluation of a number of randomly cloned BDs.…”

Gene Therapy for Skin Diseases

“…As such, short inhibitory RNAs (siRNAs) and SMarTare being investigated as methods for disease correction for EBS (Wally et al 2010;Bowden 2011;Chamcheu et al 2012). …”